The present invention relates to vaccines, preferably for cats, produced from recombinant feline herpesviruses, and to the methods for obtaining and preparing these recombinant viruses. In particular, the present invention relates more particularly to the feline herpesvirus recombinants comprising an expression cassette for one or more foreign genes.
Feline infectious rhinotracheitis is caused by feline herpesvirus type 1 (FHV-1). Feline herpesvirus (FHV-1) is classified in the Alphaherpesviridae family. Feline infectious rhinotracheitis is a disease which is very widespread in cats and, in practice, all medicated cats are vaccinated against this viral condition. There are currently several vaccines for preventing infectious rhinotracheitis. These vaccines are either of the attenuated live type, or of the inactivated type (whole virus or purified subunits). The attenuation of the live vaccines currently used has been obtained by repeated passages on cells, and the cause of their attenuation is not known. Furthermore, these vaccines exhibit, in general, a residual virulence and are for this reason administered via the parenteral (subcutaneous or intramuscular) route rather than via the intranasal route (which would nevertheless be the preferred route given the local replication of this virus). Inactivated vaccines exhibit good safety, but their weak immunogenicity requires multiple injections in order to induce a satisfactory protection.
Moreover, domestic cats are exposed to numerous other diseases, and the development of a vaccinal vector which can express various antigens of feline pathogenic agents would make it possible to simplify and improve the efficacy of vaccination programmes.
Finally, among the diseases affecting domestic cats, some are still resistant to conventional vaccinal approaches. The most well known case is that of feline infectious peritonitis caused by a coronavirus (feline infectious peritonitis (FIP) virus or FIPV).
An FHV-1 vector, whose attenuation would be such that it can be administered via the oronasal route in cats without causing local and/or general pathology, and which would allow the induction of a protective immune response both against feline infectious rhinotracheitis and against other feline pathogenic agents would constitute a significant advance in the field of vaccination of domestic feline populations.
A number of FHV genes have already been proposed as insertion sites: Patent Application EP-A-0,447,303 proposed insertion into the RR2 site of alphaherpesviruses, including FHV. The application gives the means for carrying out the insertion into the RR2 site of the turkey herpesvirus (HVT virus). Patent Application WO-A-90 01547 proposes FHV TK-vectors for the expression of heterologous genes. Likewise, Patent Application WO-A-93 09238 proposes a vaccine against feline leukaemia formed of an FHV vector in which an FHV gene has been inserted into the TK gene of the FHV virus. See also along the same lines the articles by R. C. Wardley et al. in J. Gen. Virol. 1992. 73. 1811-1818 and by G. E. Cole et al. in J. Virol. 1990. 64. 4930-4938. Patent Application WO-A-94 03621 proposes insertion into the gI, gE, US9, US10 and US11 genes. Patent Application WO-A-95 00172 proposes inserting a DNA serving as marker into the region of the genome comprising the gI and gE genes. Patent Application EP-A-0,576,092 proposes the open reading frame (ORF) situated between the gC gene and the homologous gene of the HSV-1 UL46 gene as preferential site for insertion into the FHV genome. See also M. J. Willemse et al. in J. Gen. Virol. 1994. 75. 3107-3116.
Various promoters, including those generally commercially available, have been used in the various constructs described in the prior art, among which are the HCMV IE (human CMV immediate early) promoter, the promoter sequence of the LTR region of the RSV virus (Rous Sarcoma Virus), and the SV40 virus early promoter.
The aim of the present invention is to provide a live FHV vaccine, which is attenuated but which has conserved a good capacity for replication in vivo, for immunizing cats against infectious rhinotracheitis.
Another aim of the invention is to provide a recombinant live vaccine based on FHV which is effective against other feline pathogenic agents. In particular, because of the numerous failures observed in vaccination against feline infectious peritonitis with inactivated or attenuated live vaccines, mainly because of the phenomenon of xe2x80x9cfacilitationxe2x80x9d (exacerbation of the disease), the need still exists for a vaccine which is really effective against FIP. Such a vaccine based on a recombinant FHV-1 vector which would be really effective against feline infectious peritonitis, a disease for which no one has yet marketed a satisfactory vaccine, could, in addition, pave the way for highly effective vaccines against other cat diseases such as, for example, and inter alia, feline leukaemia, feline immunodeficiency syndrome due to FIV, or feline panleukopenia.
Yet another aim of the invention is to allow an effective vaccination of cats using the oronasal route.
The applicant has characterized a new part of the FHV genome, in which it has characterized new regions of the FHV virus genome, regions called hereinafter (see Example 3) FHV ORF2 and FHV ORF5, which have proved utilizable for the insertion of foreign genes under conditions which make it possible to meet the objectives set out above.
The subject of the present invention is therefore a recombinant live vaccine using, as vector, a feline herpesvirus comprising, and expressing, at least one nucleotide sequence encoding a polypeptide, this sequence being inserted into the ORF5 site and/or ORF2 site.
Preferably, the inserted sequence encodes an antigenic polypeptide and, preferentially, an antigenic polypeptide of a feline pathogenic agent. Sequences encoding immunomodulatory proteins such as cytokines may also be inserted. According to an advantageous feature, it is possible to combine a sequence encoding a cytokine, and the like, with a sequence encoding an antigen. If necessary, several cytokine sequences may be combined with each other, optionally in combination with one or more sequences encoding antigens.
Insertion into the two sites newly characterized is carried out by simple insertion (without deletion) or after partial or total deletion of the ORFs used as insertion sites.
According to a particularly preferred feature of the invention, insertions and/or deletions are carried out in the two sites described. This feature is particularly adapted to the use of virulent wild-type strains of the FHV-1 virus for obtaining recombinants. It is therefore possible to insert at least one nucleotide sequence into each of the sites, or alternatively to insert into only one site, preferably ORF5, and to delete all or part of the other site.
According to another feature, which applies more to the attenuated vaccinal strains, without being limited thereto, the insertion is carried out in only one of the two sites, preferably the ORF5 site.
The feline herpesviruses according to the invention are preferably FHV viruses type 1.
It is possible to use, in particular, the FHV-1 CO strain in which the sequence of the genomic region (ORF1 to ORF8) is indicated in the sequence listing under the reference SEQ ID No. 1 (see also Table 1 Example 3). The ORF2 site is situated between nucleotides 1655 and 2596. The ORF5 site is situated between nucleotides 5869 and 7113.
For the expression of the foreign genes inserted into the FHV-1 genome according to the present invention, a strong eukaryotic promoter such as, preferentially, a CMV immediate early (IE) promoter will be preferably used. A CMV IE promoter is understood to mean especially a fragment as given in the example as well as its subunits such conserve the same promoter activity. The CMV IE promoter may be the human promoter (HCMV IE) or the murine promoter (MCMV IE), or alternatively a CMV IE promoter of another origin, for example from rats, guinea pigs or pigs.
It will be possible to insert at least two nucleotide sequences into one of the ORF2 or ORF5 sites under the control of different promoters. These may be especially CMV IE promoters of different origins.
According to an advantageous development of the invention, another promoter is combined with the CMV IE promoter according to a tandem arrangement, such as the 5xe2x80x2 ends of both promoters are adjacent therebetween and the transcriptions from these promoters go in opposite directions, which makes it possible to insert, into the region of insertion, two nucleotide sequences, one under the control of the CMV IE promoter, the other under that of the associated promoter. This construct is remarkable in that the presence of the CMV IE promoter, and especially of its enhancer part, may enhance the transcription induced by the associated promoter. As associated promoter, there may be mentioned, for example, a CMV promoter from a species different from the first promoter. It is also possible to envisage other promoters such as the RNA1.8 promoter from the Marek""s disease virus (MDV) (G. Bradley et al. J. Virol. 1989. 63. 2534-2542).
The nucleotide sequence inserted into the FHV vector so as to be expressed may be any sequence encoding an antigenic polypeptide of a feline pathogenic agent capable, once expressed under the favourable conditions offered by the invention, of providing immunization leading to an effective protection of the vaccinated animal against the pathogenic agent. It is therefore possible to insert, under the conditions described by the present invention, the nucleotide sequences encoding the antigens of interest for a given disease.
The typical case of the invention is the insertion of at least one nucleotide sequence suitably encoding a polypeptide of the feline infectious peritonitis virus (FIP or FIPV) and, preferably, the FIPV M poly-peptide or the modified FIPV S polypeptide. A recombinant live vaccine is thus obtained which provides, in addition to protection against feline infectious rhinotracheitis, protection against feline infectious peritonitis. If desired, a sequence encoding another antigen of the FIP virus, such as the N protein, the 7b protein and/or the polypeptides encoded by the polymerase (polB) gene of the FIP virus can also be inserted in addition or instead.
Other preferred cases of the invention are the insertion of nucleotide sequences encoding antigens or fragments of antigens of the feline leukaemia virus (FeLV), in particular env, gag and pol genes (Osterhaus A. et al. J. Immunol., 1985. 135. 591-596; Lutz H. Vet. Microbiol. 1990. 23. 131-146; Clark N. et al. JAVMA, 1991. 199. 1433-1443; Thomsen D. et al. J. Gen. Virol., 1992. 73. 1819-1824), of the feline immunodeficiency virus (FIV) (Jarrett O. et al. AIDS, 1990, 4 (suppl. 1): S163-S165; Miyazawa T. et al. Arch. Virol; 1994, 134, 221-234; de Rhonde A. et al. Virology, 1994. 198. 257-264), in particular env, gag and pol genes, of the feline panleukopenia virus (FPV) (Carlson J. et al. J. Virol. 1985. 55. 574-582; Martyn J. et al. J. Gen. Virol. 1990. 71, 2747-2753), in particular the VP2 capsid gene, of the feline calicivirus (FCV) (Neill J. et al. J. Virol. 1991. 65. 5440-5447; Carter M. et al. Virology. 1992. 190 443-448), in particular the capsid gene.
A typical case of the invention is a vaccine comprising a nucleotide sequence encoding an antigen of the FIP virus under the control of CMV IE and a nucleotide sequence encoding an antigen of another feline viral disease, especially those mentioned above, under the control of another promoter.
The subject of the present invention is also a polyvalent vaccine formula comprising, in the form of a mixture or to be mixed, at least two recombinant live vaccines as defined above, these vaccines comprising different inserted sequences, especially from different pathogens.
The subject of the present invention is also the FHV viruses modified in one or both ORF2 and ORF5 sites as indicated above.
Its subject is also a method of vaccination, in particular of cats, in which an effective quantity of a vaccine as defined above is administered by any parenteral or local route, but preferably by the oronasal route. The vaccinal dose will be between 102 CCID50 and 107 CCID50. As defined, the vaccine is effective in general after only one administration by the oronasal route. However, repeated administrations may be necessary.
The subject of the present invention is also the DNA fragments comprising all or part of the sequence defined by positions 1 to 8193 on SEQ ID No. 1, especially all or part of the ORF2 and ORF5 sites defined and/or of the flanking sequences located upstream and downstream of these sites, which fragments will be useful as flanking arms for the techniques of homologous recombination with the genome of the parenteral FHV virus. Of course, the invention also relates to the variants of these fragments which correspond to the equivalent sequences of the other FHV strains. The specialist is quite free to choose the regions serving as flanking arms in conjunction with the type of insertion (with or without deletion) or deletion (partial or total) chosen. In general, the flanking arms can thus have from 100 to 800 base pairs.